To examine the role of sphingosine 1-phosphate (S1P) receptor 3 (S1P₃) in modulating muscle properties, we utilized transgenic mice depleted of the receptor. Morphological analyses of extensor digitorum longus (EDL) muscle did not show evident differences between wild-type and S1P₃-null mice. The body weight of 3-mo-old S1P₃-null mice and the mean cross-sectional area of transgenic EDL muscle fibers were similar to those of wild-type. S1P₃deficiency enhanced the expression level of S1P₁and S1P₂receptors mRNA in S1P₃-null EDL muscle. The contractile properties of S1P₃-null EDL diverge from those of wild-type, largely more fatigable and less able to recover. The absence of S1P₃appears responsible for a lower availability of calcium during fatigue. S1P supplementation, expected to stimulate residual S1P receptors and signaling, reduced fatigue development of S1P₃-null muscle. Moreover, in the absence of S1P₃, denervated EDL atrophies less than wild-type. The analysis of atrophy-related proteins in S1P₃-null EDL evidences high levels of the endogenous regulator of mitochondria biogenesis peroxisome proliferative-activated receptor-γ coactivator 1α (PGC-1α); preserving mitochondria could protect the muscle from disuse atrophy. In conclusion, the absence of S1P₃makes the muscle more sensitive to fatigue and slows down atrophy development after denervation, indicating that S1P₃is involved in the modulation of key physiological properties of the fast-twitch EDL muscle.